Multiplexed Opto-Microfluidic Biosensing: Advanced Platform for Prostate Cancer Detection.

Cancer stands as a prominent global cause of mortality, necessitating early detection to augment survival rates and alleviate economic burdens on healthcare systems. In particular, prostate cancer (PCa), impacting 1.41 million men globally in 2020, accentuates the demand for sensitive and cost-effective detection methods beyond traditional prostate-specific antigen (PSA) testing. While clinical techniques exhibit limitations, biosensors emerge as compact, user-friendly alternatives to traditional laboratory approaches. However, existing biosensors predominantly concentrate on PSA detection, prompting the necessity for advancing toward multiplex sensing platforms. This study introduces a compact opto-microfluidic sensor featuring a substrate of gold nanospikes, fabricated via electrodeposition, for enhanced sensitivity. Embedded within a microfluidic chip, this nanomaterial enables the precise and concurrent measurement of PSA, alongside two complementary PCa biomarkers, matrix metalloproteinase-2 (MMP-2) and anti-α-methylacyl-CoA racemase (anti-AMACR) in diluted human plasma, offering a comprehensive approach to PSA analysis. Taking advantage of the localized surface plasmon resonance principle, this biosensor offers robustness and sensitivity in real sample analysis without the need for labeling agents. With the limit of detection at 0.22, 0.37, and 0.18 ng/mL for PSA, MMP-2, and anti-AMACR, respectively, this biosensing platform holds promise for point-of-care analysis, underscoring its potential impact on medical diagnostics.

Nonclinical Investigation of Cytokine Mitigation Strategies for T-cell-Engaging Bispecifics in the Cynomolgus Macaque.

SUMMARY: T-cell-directed cancer therapies such as T-cell-engaging bispecifics (TCBs) are commonly associated with cytokine release syndrome and associated clinical signs that can limit their tolerability and therapeutic benefit. Strategies for reducing cytokine release are therefore needed. Here, we report on studies performed in cynomolgus monkeys to test different approaches for mitigating cytokine release with TCBs. A "priming dose" as well as subcutaneous dosing reduced cytokine release compared with intravenous dosing but did not affect the intended T-cell response to the bispecific. As another strategy, cytokines or cytokine responses were blocked with an anti-IL-6 antibody, dexamethasone, or a JAK1/TYK2-selective inhibitor, and the effects on toxicity as well as T-cell responses to a TCB were evaluated. The JAK1/TYK2 inhibitor and dexamethasone prevented CRS-associated clinical signs on the day of TCB administration, but the anti-IL-6 had little effect. All interventions allowed for functional T-cell responses and expected damage to target-bearing tissues, but the JAK1/TYK2 inhibitor prevented the upregulation of activation markers on T cells, suggesting the potential for suppression of T-cell responses. Our results suggest that short-term prophylactic dexamethasone treatment may be an effective option for blocking cytokine responses without affecting desired T-cell responses to TCBs.

Recombinant adeno-associated virus production evaluation in Chinese hamster ovary cells.

The gene therapy field has advanced in recent years with five recombinant adeno-associated virus (rAAV) based products winning Food and Drug Administration (FDA) approval. As the number of therapeutic applications and overall production demands for rAAV increase, it is valuable to evaluate rAAV production in different production cells. Chinese hamster ovary (CHO) cells have been a robust host for biomolecule manufacturing for more than 35 years. However, there is no report to our knowledge describing the use of CHO cells for rAAV production. In this study, we examined the ability of CHO cells to produce rAAV using a transient plasmid transfection approach. Our results demonstrated that CHO is capable of producing rAAV with detectable viral fundamental components including viral RNAs, proteins, and rAAV viral particles. We identified the expression of cap proteins as one of the limiting factors for rAAV production in CHO cells. We therefore added an additional cytomegalovirus (CMV)-Cap plasmid to the CHO transfection. After increasing cap protein expression, we detected rAAV titers as high as 3 × 108 viral genomes for every 2 × 109 capsids in CHO cells using a quintuple transfection method (standard AAV2 Rep/Cap, helper, gene of interest plasmids, plus CMV-E1, and CMV-Cap plasmids) with comparable full particle percent (average 15%) to that of human embryo kidney (HEK)-derived rAAV. Our study provides a foundation for potential rAAV production in CHO cells.

Case report: Successful treatment with tofacitinib and colchicine in a patient with Schnitzler syndrome.

We report a case of late-onset Schnitzler syndrome successfully treated with Janus-activated kinase (JAK) inhibitors and colchicine. Schnitzler syndrome should be considered for recurrent chronic urticaria when accompanied by fever, fatigue, rapid weight loss, and poor response to antihistamine treatment. Skin biopsy, bone marrow biopsy, and electrophoresis help confirm the diagnosis. Early diagnosis and treatment can lead to complete resolution of symptoms. Besides interleukin (IL)-1 and IL-6 inhibitors, JAK inhibitors and colchicine may be considered as other choices of treatment.

Voltage-gated ion channels mediate the electrotaxis of glioblastoma cells in a hybrid PMMA/PDMS microdevice.

Transformed astrocytes in the most aggressive form cause glioblastoma, the most common cancer in the central nervous system with high mortality. The physiological electric field by neuronal local field potentials and tissue polarity may guide the infiltration of glioblastoma cells through the electrotaxis process. However, microenvironments with multiplex gradients are difficult to create. In this work, we have developed a hybrid microfluidic platform to study glioblastoma electrotaxis in controlled microenvironments with high throughput quantitative analysis by machine learning-powered single cell tracking software. By equalizing the hydrostatic pressure difference between inlets and outlets of the microchannel, uniform single cells can be seeded reliably inside the microdevice. The electrotaxis of two glioblastoma models, T98G and U-251MG, requires an optimal laminin-containing extracellular matrix and exhibits opposite directional and electro-alignment tendencies. Calcium signaling is a key contributor in glioblastoma pathophysiology but its role in glioblastoma electrotaxis is still an open question. Anodal T98G electrotaxis and cathodal U-251MG electrotaxis require the presence of extracellular calcium cations. U-251MG electrotaxis is dependent on the P/Q-type voltage-gated calcium channel (VGCC) and T98G is dependent on the R-type VGCC. U-251MG electrotaxis and T98G electrotaxis are also mediated by A-type (rapidly inactivating) voltage-gated potassium channels and acid-sensing sodium channels. The involvement of multiple ion channels suggests that the glioblastoma electrotaxis is complex and patient-specific ion channel expression can be critical to develop personalized therapeutics to fight against cancer metastasis. The hybrid microfluidic design and machine learning-powered single cell analysis provide a simple and flexible platform for quantitative investigation of complicated biological systems.

Intracellular Nanomaterial Delivery via Spiral Hydroporation.

In recent nanobiotechnology developments, a wide variety of functional nanomaterials and engineered biomolecules have been created, and these have numerous applications in cell biology. For these nanomaterials to fulfill their promises completely, they must be able to reach their biological targets at the subcellular level and with a high level of specificity. Traditionally, either nanocarrier- or membrane disruption-based method has been used to deliver nanomaterials inside cells; however, these methods are suboptimal due to their toxicity, inconsistent delivery, and low throughput, and they are also labor intensive and time-consuming, highlighting the need for development of a next-generation, intracellular delivery system. This study reports on the development of an intracellular nanomaterial delivery platform, based on unexpected cell-deformation phenomena via spiral vortex and vortex breakdown exerted in the cross- and T-junctions at moderate Reynolds numbers. These vortex-induced cell deformation and sequential restoration processes open cell membranes transiently, allowing effective and robust intracellular delivery of nanomaterials in a single step without the aid of carriers or external apparatus. By using the platform described here (termed spiral hydroporator), we demonstrate the delivery of different nanomaterials, including gold nanoparticles (200 nm diameter), functional mesoporous silica nanoparticles (150 nm diameter), dextran (hydrodynamic diameters between 2-55 nm), and mRNA, into different cell types. We demonstrate here that the system is highly efficient (up to 96.5%) with high throughput (up to 1 × 106 cells/min) and rapid delivery (∼1 min) while maintaining high levels of cell viability (up to 94%).

Glioblastoma adhesion in a quick-fit hybrid microdevice.

Translational research requires reliable biomedical microdevices (BMMD) to mimic physiological conditions and answer biological questions. In this work, we introduce a reversibly sealed quick-fit hybrid BMMD that is operator-friendly and bubble-free, requires low reagent and cell consumption, enables robust and high throughput performance for biomedical experiments. Specifically, we fabricate a quick-fit poly(methyl methacrylate) and poly(dimethyl siloxane) (PMMA/PDMS) prototype to illustrate its utilities by probing the adhesion of glioblastoma cells (T98G and U251MG) to primary endothelial cells. In static condition, we confirm that angiopoietin-Tie2 signaling increases the adhesion of glioblastoma cells to endothelial cells. Next, to mimic the physiological hemodynamic flow and investigate the effect of physiological electric field, the endothelial cells are pre-conditioned with concurrent shear flow (with fixed 1 Pa shear stress) and direct current electric field (dcEF) in the quick-fit PMMA/PDMS BMMD. With shear flow alone, endothelial cells exhibit classical parallel alignment; while under a concurrent dcEF, the cells align perpendicularly to the electric current when the dcEF is greater than 154 V m- 1. Moreover, with fixed shear stress of 1 Pa, T98G glioblastoma cells demonstrate increased adhesion to endothelial cells conditioned in dcEF of 154 V m- 1, while U251MG glioblastoma cells show no difference. The quick-fit hybrid BMMD provides a simple and flexible platform to create multiplex systems, making it possible to investigate complicated biological conditions for translational research.

Microfluidic Assisted Nanoprecipitation of PLGA Nanoparticles for Curcumin Delivery to Leukemia Jurkat Cells.

The ability to control particle size and size distribution of nanoparticles for drug delivery is essential because it impacts on the biodistribution and cellular uptake of nanoparticles. We present a novel microfluidic assisted nanoprecipitation strategy that enables synthesis of surfactant-free curcumin encapsulated poly(lactide- co-glycolide) nanoparticles (Cur-PLGA NP) with adjustable particle diameters (30-70 nm) and narrow particle size distribution (polydispersity index less than 0.2). Our Cur-PLGA NP exhibit excellent colloidal stability and inhibit degradation of curcumin. We further demonstrate the potential of our Cur-PLGA NP as a nanotoxic delivery system for curcumin. Cellular viability assay validates a dose-dependent cytotoxicity of Cur-PLGA NP in leukemia Jurkat cells. In contrast, Cur-PLGA NP does not alter the viability of fibroblast NIH3T3 cells, which suggests that the cytotoxicity of Cur-PLGA NP is specific to cell types. Furthermore, there is no detectable effect by PLGA NP to both leukemia Jurkat cells and fibroblast NIH3T3 cells, highlighting the nontoxic nature of our delivery system. Confocal cell uptake studies indicate that PLGA NP do not alter the cell uptake of curcumin. Our microfluidic assisted approach offers a controlled and effective nanobiomaterials synthesis of drug delivery system for curcumin, which can be extended to different capsule materials for a variety of biomedical applications.

Microfluidic device flow field characterization around tumor spheroids with tunable necrosis produced in an optimized off-chip process.

Tumor spheroids are a 3-D tumor model that holds promise for testing cancer therapies in vitro using microfluidic devices. Tailoring the properties of a tumor spheroid is critical for evaluating therapies over a broad range of possible indications. Using human colon cancer cells (HCT-116), we demonstrate controlled tumor spheroid growth rates by varying the number of cells initially seeded into microwell chambers. The presence of a necrotic core in the spheroids could be controlled by changing the glucose concentration of the incubation medium. This manipulation had no effect on the size of the tumor spheroids or hypoxia in the spheroid core, which has been predicted by a mathematical model in computer simulations of spheroid growth. Control over the presence of a necrotic core while maintaining other physical parameters of the spheroid presents an opportunity to assess the impact of core necrosis on therapy efficacy. Using micro-particle imaging velocimetry (micro-PIV), we characterize the hydrodynamics and mass transport of nanoparticles in tumor spheroids in a microfluidic device. We observe a geometrical dependence on the flow rate experienced by the tumor spheroid in the device, such that the "front" of the spheroid experiences a higher flow velocity than the "back" of the spheroid. Using fluorescent nanoparticles, we demonstrate a heterogeneous accumulation of nanoparticles at the tumor interface that correlates with the observed flow velocities. The penetration depth of these nanoparticles into the tumor spheroid depends on nanoparticle diameter, consistent with reports in the literature.

Tumour-on-a-chip: microfluidic models of tumour morphology, growth and microenvironment.

Cancer remains one of the leading causes of death, albeit enormous efforts to cure the disease. To overcome the major challenges in cancer therapy, we need to have a better understanding of the tumour microenvironment (TME), as well as a more effective means to screen anti-cancer drug leads; both can be achieved using advanced technologies, including the emerging tumour-on-a-chip technology. Here, we review the recent development of the tumour-on-a-chip technology, which integrates microfluidics, microfabrication, tissue engineering and biomaterials research, and offers new opportunities for building and applying functional three-dimensional in vitro human tumour models for oncology research, immunotherapy studies and drug screening. In particular, tumour-on-a-chip microdevices allow well-controlled microscopic studies of the interaction among tumour cells, immune cells and cells in the TME, of which simple tissue cultures and animal models are not amenable to do. The challenges in developing the next-generation tumour-on-a-chip technology are also discussed.

Dynamics of a Water Droplet over a Sessile Oil Droplet: Compound Droplets Satisfying a Neumann Condition.

We report the dynamics of compound droplets with a denser liquid (water) droplet over a less dense sessile droplet (mineral oil) that satisfies the Neumann condition. For a fixed size of an oil droplet, depending on the size of the water droplet, either it attains the axisymmetric position or tends to migrate toward the edge of the oil droplet. For a water droplet-to-oil droplet at volume ratio Vw/Vo ≥ 0.05, stable axisymmetric configuration is achieved; for Vw/Vo < 0.05, migration of water droplet is observed. The stability and migration of water droplets of size above and below critical size, respectively, are explained using the force balance at the three-phase contact line and film tension. The larger and smaller droplets that initially attain the axisymmetric position or some radial position, respectively, evaporate continuously and thus migrate toward the edge of the oil droplet. The radial location and migration of the water droplets of different initial sizes with respect to time are studied. Experiments with water droplets on a flat oil-air interface did not show migration, which signified the role of the curved oil-air interface for droplet migration. Finally, coalescence of water droplets of size above the critical size at the axisymmetric position is demonstrated. Our compound droplet studies could be beneficial for applications involving droplet transport where contamination due to direct contact and pinning of droplets on solid surfaces is of concern. Migration and coalescence of water droplets on curved oil-air interfaces could open new frontiers in chemical and biological applications including multiphase processing and biological interaction of cells and atmospheric chemistry.

Microcontact printing with aminosilanes: creating biomolecule micro- and nanoarrays for multiplexed microfluidic bioassays.

Microfluidic systems integrated with protein and DNA micro- and nanoarrays have been the most sought-after technologies to satisfy the growing demand for high-throughput disease diagnostics. As the sensitivity of these systems relies on the bio-functionalities of the patterned recognition biomolecules, the primary concern has been to develop simple technologies that enable biomolecule immobilization within microfluidic devices whilst preserving bio-functionalities. To address this concern, we introduce a two-step patterning approach to create micro- and nanoarrays of biomolecules within microfluidic devices. First, we introduce a simple aqueous based microcontact printing (µCP) method to pattern arrays of (3-aminopropyl)triethoxysilane (APTES) on glass substrates, with feature sizes ranging from a few hundred microns down to 200 nm (for the first time). Next, these substrates are integrated with microfluidic channels to then covalently couple DNA aptamers and antibodies with the micro- and nanopatterned APTES. As these biomolecules are covalently tethered to the device substrates, the resulting bonds enable them to withstand the high shear stresses originating from the flow in these devices. We further demonstrated the flexibility of this technique, by immobilizing multiple proteins onto these APTES-patterned substrates using liquid-dispensing robots to create multiple microarrays. Next, to validate the functionalities of these microfluidic biomolecule microarrays, we perform (i) aptamer-based sandwich immunoassays to detect human interleukin 6 (IL6); and (ii) antibody-based sandwich immunoassays to detect human c-reactive protein (hCRP) with the limit of detection at 5 nM, a level below the range required for clinical screening. Lastly, the shelf-life potential of these ready-to-use microfluidic microarray devices is validated by effectively functionalizing the patterns with biomolecules up to 3 months post-printing. In summary, with a single printing step, this aminosilane patterning technique enables the creation of functional microfluidic micro- and nano-biomolecule arrays, laying the foundation for high-throughput multiplexed bioassays.

An efficient route to bispecific antibody production using single-reactor mammalian co-culture.

Bispecific antibodies have shown promise in the clinic as medicines with novel mechanisms of action. Lack of efficient production of bispecific IgGs, however, has limited their rapid advancement. Here, we describe a single-reactor process using mammalian cell co-culture production to efficiently produce a bispecific IgG with 4 distinct polypeptide chains without the need for parallel processing of each half-antibody or additional framework mutations. This method resembles a conventional process, and the quality and yield of the monoclonal antibodies are equal to those produced using parallel processing methods. We demonstrate the application of the approach to diverse bispecific antibodies, and its suitability for production of a tissue specific molecule targeting fibroblast growth factor receptor 1 and klotho ß that is being developed for type 2 diabetes and other obesity-linked disorders.

Contribution of Antibody Hydrodynamic Size to Vitreal Clearance Revealed through Rabbit Studies Using a Species-Matched Fab.

We have developed a tool Fab fragment of a rabbit monoclonal antibody that is useful for early evaluation in rabbit models of technologies for long acting delivery (LAD) of proteins to the eye. Using this Fab we show that vitreal clearance can be slowed through increased hydrodynamic size. Fab (G10rabFab) and Fab' (G10rabFab') fragments of a rabbit monoclonal antibody (G10rabIgG) were expressed in Chinese hamster ovary (CHO) cells and purified using antigen-based affinity chromatography. G10rabFab retains antigen-binding upon thermal stress (37 °C) for 8 weeks in phosphate-buffered saline (PBS) and can be detected in rabbit tissues using an antigen-based ELISA. Hydrodynamic radius, measured using quasi-elastic light scattering (QELS), was increased through site-specific modification of the G10rabFab' free cysteine with linear methoxy-polyethylene glycol(PEG)-maleimide of 20000 or 40000 molecular weight. Pharmacokinetic studies upon intravitreal dosing in New Zealand white rabbits were conducted on the G10rabFab and PEGylated G10rabFab'. Results of single and multidose pharmacokinetic experiments yield reproducible results and a vitreal half-life for G10rabFab of 3.2 days. Clearance from the eye is slowed through increased hydrodynamic size, with vitreal half-life showing a linear dependence on hydrodynamic radius (RH). A linear dependence of vitreal half-life on RH suggests that molecule diffusivity makes an important contribution to vitreal clearance. A method for prediction of vitreal half-life from RH measurements is proposed.

Sensing and Sensibility: Single-Islet-based Quality Control Assay of Cryopreserved Pancreatic Islets with Functionalized Hydrogel Microcapsules.

Despite decades of research and clinical studies of islet transplantations, finding simple yet reliable islet quality assays that correlate accurately with in vivo potency is still a major challenge, especially for real-time and single-islet-based quality assessment. Herein, proof-of-concept studies of a cryopreserved microcapsule-based quality control assays are presented for single islets. Individual rat pancreatic islets and fluorescent oxygen-sensitive dye (FOSD) are encapsulated in alginate hydrogel microcapsules via a microfluidic device. To test the susceptibility of the microcapsules and the FOSD to cryopreservation, the islet microcapsules containing FOSD are cryopreserved and the islet functionalities (adenosine triphosphate, static insulin release measurement, and oxygen consumption rate) are assessed after freezing and thawing steps. The cryopreserved islet capsules with FOSD remain functional after encapsulation and freezing/thawing procedures, validating a simple yet reliable individual-islet-based quality control method for the entire islet processing procedure prior to transplantation. This work also demonstrates that the functionality of cryopreserved islets can be improved by introducing trehalose into the routinely used cryoprotectant dimethyl sulfoxide. The functionalized alginate hydrogel microcapsules with embedded FOSD and optimized cryopreservation protocol presented in this work serve as a versatile islet quality assay and offer tremendous promise for tackling existing challenges in islet transplantation procedures.

Pharmacists' views on and experiences with bowel cancer screening kits in Auckland, New Zealand.

OBJECTIVES: To explore the views of New Zealand pharmacists on bowel cancer screening, particularly with regards to faecal occult blood testing (FOBT) kits, self-perceived knowledge on FOBT kits and barriers, motivators and experiences with selling and counselling consumers with respect to FOBT kits. METHODS: Semi-structured interviews were conducted face to face or by telephone with 20 community pharmacists in the Auckland region. Interviews were recorded and transcribed verbatim and data were coded and analysed using NVivo software to identify key themes. KEY FINDINGS: Participant pharmacists believed that they were well placed to provide advice on FOBT kits to consumers. Barriers to selling the kits included cost and perceived lack of test sensitivity of the kits, poor consumer demand, pharmacists' lack of training and information, and a belief that selling FOBT kits was outside the pharmacists' scope of practice. Motivators to selling the kits included customer convenience, ease of use, confidence in the kits and embracing new roles for pharmacists. Pharmacists were concerned that use of the kits may increase the burden on the public health system through customer anxiety over test results; however, they agreed that there was a need for bowel cancer screening and awareness and that people concerned about bowel cancer should make visiting their general practitioner a priority. CONCLUSIONS: Pharmacists' views were mixed. Pharmacists' training and competence with respect to the provision of bowel cancer kits, and how a bowel cancer screening service can be developed to optimise public health outcomes, need to be addressed.

Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity.

4-1BB (CD137, TNFRSF9) is a costimulatory receptor expressed on several subsets of activated immune cells. Numerous studies of mouse and human T cells indicate that 4-1BB promotes cellular proliferation, survival, and cytokine production. 4-1BB agonist mAbs have demonstrated efficacy in prophylactic and therapeutic settings in both monotherapy and combination therapy tumor models and have established durable anti-tumor protective T-cell memory responses. PF-05082566 is a fully human IgG2 that binds to the extracellular domain of human 4-1BB with high affinity and specificity. In preclinical studies, this agonist antibody demonstrated its ability to activate NF-κB and induce downstream cytokine production, promote leukocyte proliferation, and inhibit tumor growth in a human PBMC xenograft tumor model. The mechanism of action and robust anti-tumor efficacy of PF-05082566 support its clinical development for the treatment of a broad spectrum of human malignancies.

Decreasing lactate level and increasing antibody production in Chinese Hamster Ovary cells (CHO) by reducing the expression of lactate dehydrogenase and pyruvate dehydrogenase kinases.

Large-scale fed-batch cell culture processes of CHO cells are the standard platform for the clinical and commercial production of monoclonal antibodies. Lactate is one of the major by-products of CHO fed-batch culture. In pH-controlled bioreactors, accumulation of high levels of lactate is accompanied by high osmolality due to the addition of base to control pH of the cell culture medium, potentially leading to lower cell growth and lower therapeutic protein production during manufacturing. Lactate dehydrogenase (LDH) is an enzyme that catalyzes the conversion of the substrate, pyruvate, into lactate and many factors including pyruvate concentration modulate LDH activity. Alternately, pyruvate can be converted to acetyl-CoA by pyruvate dehydrogenases (PDHs), to be metabolized in the TCA cycle. PDH activity is inhibited when phosphorylated by pyruvate dehydrogenase kinases (PDHKs). In this study, we knocked down the gene expression of lactate dehydrogenase A (LDHa) and PDHKs to investigate the effect on lactate metabolism and protein production. We found that LDHa and PDHKs can be successfully downregulated simultaneously using a single targeting vector carrying small inhibitory RNAs (siRNA) for LDHa and PDHKs. Moreover, our fed-batch shake flask evaluation data using siRNA-mediated LDHa/PDHKs knockdown clones showed that downregulating LDHa and PDHKs in CHO cells expressing a therapeutic monoclonal antibody reduced lactate production, increased specific productivity and volumetric antibody production by approximately 90%, 75% and 68%, respectively, without appreciable impact on cell growth. Similar trends of lower lactate level and higher antibody productivity on average in siRNA clones were also observed from evaluations performed in bioreactors.

Identification of IgG(1) variants with increased affinity to FcγRIIIa and unaltered affinity to FcγRI and FcRn: comparison of soluble receptor-based and cell-based binding assays.

Clinical response to the anti-CD20 antibody rituximab has been demonstrated to correlate with the polymorphism in the FcγRIIIa receptor where patients homozygous for the higher affinity V158 allotype showed a better response rate. This finding suggests that engineering of anti-CD20 for increased FcγRIIIa affinity could result in improved clinical outcome. To identify variants with increased affinity to FcγRIIIa, we developed quantitative assays using soluble receptors as well as engineered cell lines expressing FcγRI or FcγRIIIa on the cell surface. We assayed a set of anti-CD20 IgG(1) variants that had identical Fab regions, but alterations in the Fc regions, in both the soluble receptor-based and cell-based FcγRIIIa binding assays. We obtained similar relative binding affinity increases and assay precisions. The increase in affinity for FcγRIIIa correlated with the increase in activity in the antibody-dependent cellular cytotoxicity assay. These variants had unaltered FcγRI binding. In addition to Fcγ receptors, IgG also binds to FcRn, the receptor responsible for the long circulating half-life of IgG. The mutations in the anti-CD20 variants were previously found not to affect FcRn binding in the soluble receptor-based assays; consequently, we used anti-Her2 variants with different binding affinities to FcRn to study FcRn binding assays. We generated a cell line expressing FcRn on the cell surface to measure IgG binding and obtained similar ranking of these anti-Her2 variants in the cell-based and the soluble receptor-based FcRn binding assays. In conclusion, both the soluble receptor-based and cell-based binding assays can be used to identify IgG(1) variants with increased affinity to FcγRIIIa and unaltered affinity to FcγRI and FcRn.